The Involvement of Protein Phosphatases in the Activation of ICE/CED-3 Protease, Intracellular Acidification, DNA Digestion, and Apoptosis

Abstract

Many events in apoptosis have been identified but their temporal relationships remain obscure. Apoptosis in human ML-1 cells induced by etoposide is characterized by intracellular acidification, enhanced Hoechst 33342 fluorescence, DNA digestion, chromatin condensation, and proteolysis of poly(ADP-ribose) polymerase. This proteolysis is a marker for the action of ICE/CED-3 proteases, which are critical activators of apoptosis. We observed that three serine/threonine protein phosphatase inhibitors, okadaic acid, calyculin A, and cantharidin, prevented all of these apoptotic characteristics. To determine which protein phosphatase was involved, we investigated the dephosphorylation of the retinoblastoma susceptibility protein Rb, a substrate for protein phosphatase 1 but not protein phosphatase 2A. Rb was dephosphorylated during apoptosis, and each inhibitor prevented this dephosphorylation at the same concentrations that prevented apoptosis. No increase in protein phosphatase 1 activity was observed in apoptotic cells suggesting that dephosphorylation of Rb may result from loss of Rb kinase activity in the presence of a constant level of protein phosphatase activity. Long term inhibition of protein phosphatase 1 (>8 h) also led to the appearance of dephosphorylated Rb, cleavage of poly(ADP-ribose) polymerase and apoptosis, suggesting these events are not solely dependent upon protein phosphatase 1. Rb dephosphorylation was also observed in several other models of apoptosis. Hence, an imbalance between protein phosphatase 1 and Rb kinase may be a common means to activate ICE/CED-3 proteases resulting in the subsequent events of apoptosis.Maintaining tissue and organ homeostasis requires a delicate balance between the rate of cell division and the rate of cell death. Cells that are either metabolically compromised or no longer required are eliminated by a pathway commonly known as apoptosis. This process has been characterized by cell shrinkage, cytoplasmic blebbing, chromosome condensation, DNA digestion, and, finally, non-inflammatory removal of the cell from the tissue (1). Apoptosis occurs during development, immune regulation, and normal cell turnover, as well as being induced by many pharmacological insults. Decreased apoptosis can lead to cancer and autoimmune diseases, while increased apoptosis can result in neurodegenerative disorders and AIDS (2). Despite the importance of apoptosis, the signal transduction pathways responsible for the associated morphological and biochemical changes are poorly understood. Much recent emphasis has focused on the identification of proteases of the ICE/CED-3 family that appear essential to this pathway (3-5), but both the upstream and downstream events remain elusive.While investigating endonucleases that might be involved in apoptosis, we detected and purified deoxyribonuclease II (DNase II), an endonuclease that is active at slightly acidic pH but inactive at pH 7.0 (6). To determine whether DNase II might be responsible for the DNA digestion, we measured intracellular pH ...

Susceptibility of HIV-1-TAT transfected cells to undergo apoptosis. Biochemical mechanisms

“…Although the phosphorylation status of Bad in K562 and K562-Tat cells remain to be investigated, it is tempting to speculate that Tat localized in the mitochondria may activate some phosphatases that mediate Bad dephosphorylation, then allowing to form heterodimers with Bcl-X L to exert its apoptotic function (Gajewski and Thompson, 1996;Zha et al, 1996). In this sense, the role of serine phosphatases in the process of apoptosis has been also suggested (Morana et al, 1996). Alternatively, Tat may inhibit the activity of the kinase Raf-1, which has been shown to phosphorylate Bad inhibiting its pro-apoptotic function (Wang et al, 1996).…”

Zinc inhibits apoptosis upstream of ICE/CED-3 proteases rather than at the level of an endonuclease

“…Human ML-1 cells show chromatin condensation, DNA fragmentation and intracellular acidification within 4 h after a 30 min incubation with the topoisomerase II inhibitor etoposide (Morana et al, 1994(Morana et al, , 1996. This time period has been used for the studies reported here.…”

“…The intracellular activity of this protease family can also be assessed by the cleavage of an endogenous protein, poly(ADP-ribose) polymerase (PARP) (Lazebnik et al, 1994). We have recently established a temporal order of events in apoptosis in which dephosphorylation of the retinoblastoma susceptibility protein Rb occurs upstream of cleavage of PARP, DNA digestion and apoptosis (Morana et al, 1996). In the current experiments, we show that zinc prevents all of these events, demonstrating that the protective action of zinc is upstream of both Rb dephosphorylation and activation of ICE/CED-3 proteases and not at the level of an endonuclease.…”

Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities

“…PP-1 promotes survival through regulation of p53 and other targets It has been well documented that inhibition of PP-1 or/and PP-2A by the marine algae toxins, okadaic acid and calyculin A, induces apoptosis of various types of cells (Boe et al, 1991;Ishida et al, 1992;Mellgren et al, 1993;Kiguchi et al, 1994;Weller et al, 1995;Fernandez-Sanchez et al, 1996;Morana et al, 1996;Sheikh et al, 1996;Yan et al, 1997;Li et al, 1998Li et al, , 2001aGarcia et al, 2003). However, why inhibition of PP-1 or/and PP-2A leads to apoptosis remains largely unknown.…”